US20110076240A1 - Antifungal and Anti-Cariogenic Cellobio-Oligosaccharides Produced by Dextransucrase - Google Patents

Antifungal and Anti-Cariogenic Cellobio-Oligosaccharides Produced by Dextransucrase Download PDF

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US20110076240A1
US20110076240A1 US12/988,343 US98834309A US2011076240A1 US 20110076240 A1 US20110076240 A1 US 20110076240A1 US 98834309 A US98834309 A US 98834309A US 2011076240 A1 US2011076240 A1 US 2011076240A1
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oligosaccharides
cellobio
glucopyranosyl
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cellobiose
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Donal F. Day
Misook Kim
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Louisiana State University and Agricultural and Mechanical College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics

Definitions

  • This invention relates to compositions and methods using cellobio-oligosaccharides to inhibit caries formation by preventing the production of bacterial biofilms on teeth and to inhibit growth of fungi, e.g., Aspergillus , in part by inhibiting ⁇ -1,3 glucan synthase, an enzyme essential for fungal cell wall formation.
  • fungi e.g., Aspergillus
  • Oligosaccharides are carbohydrate polymers, generally with two to ten monomeric residues linked by O-glycosidic bonds. Oligosaccharides have been widely used in food, animal feed, pharmaceutical, and cosmetic industries for a long time due to their beneficial effects on human and animals (Eggleston and Côte, 2003). Most commercial oligosaccharides were originally developed as sweeteners, but currently are valued more as soluble fibers, which decrease gastrointestinal transit time and moderate constipation and diarrhea. Oligosaccharides are considered to be a low calorie food, because they are resistant to attack by digestive enzymes in human and animals and are not absorbed by the host.
  • Oligosaccharides may be produced through microbial fermentation, enzymatic synthesis, or extraction from naturally occurring sources.
  • the major commercial oligosaccharides include cyclomaltodextrins, maltodextrins, fructooligosaccharides, galactooligosaccharides, and soy oligosaccharides (Eggleston and Côte, 2003).
  • Various oligosaccharides have been investigated for physiological functions including immune-stimulating, anti-cariogenic, and prebiotic compounds as well as sweeteners, stabilizers, and bulking agents (Eggleston and Côte, 2003; Otaka, 2006). Most of the beneficial effects related to health are reported to be as inhibitors against enzymes involved in carbohydrate metabolism.
  • Enzymatic production of oligosaccharides has many advantages over other methods, especially chemical methods. In most cases, the enzymatic synthesis of oligosaccharides has used transglycosylation reactions between a specific donor and a relatively large variety of structurally different acceptors. The configuration of the glycosidic bond produced is a function of the specificity of the transfer by the specific enzyme (Fu et al., 1990; Robyt, 1995). Various oligosaccharides have been produced by enzymatic transfer reactions from glucansucrases, amylosucrases, cyclodextrin glucanosyltransferases, and sialy transferases.
  • Dextransucrase (EC 2.4.5.1) is a glucosyltransferase, which catalyses the transfer of D-glucopyranosyl residues from sucrose to dextran. It catalyzes the synthesis of a dextran containing 50% or more ⁇ -1,6 glucosidic bonds in the main chain. However, in the presence of an alternate efficient acceptor molecule, its action changes to produce oligosaccharides.
  • Cellobiose is a disaccharide composed of two glucose molecules linked with a ⁇ -1,4 bond, which is produced during the enzymatic hydrolysis of cellulose. Another class of sugars containing cellobiose as a component are produced by transglycosylation reactions (Lee et al., 2003; Morales et al., 2001).
  • Acarbose analogues containing cellobiose were prepared by the reaction of acarbose and cellobiose with Bacillus stearothermophilus alpha maltogenic amylase. Cellobiose-acarbose analogues inhibited ⁇ -glucosidase, whereas acarbose did not.
  • Oligosaccharides with branched chains were produced in a reaction catalyzed by alternansucrase from Leuconostoc mesenteroides NRRL B-23192 (Morales et al. (2001).
  • Leuconostoc mesenteroides B512 FMCM produces an extracellular dextransucrase which synthesizes a dextran that has 95% ⁇ -(1 ⁇ 6) linear and 5% ⁇ -(1 ⁇ 3) branched linkages, and can transfer glucosyl units from sucrose onto an acceptor to produce oligosaccharides (Lindberg and Svensson, 1968.).
  • ⁇ -D-glucopyranosyl-(1 ⁇ 3)- ⁇ -D-glucopyranosyl-(1 ⁇ 6)- ⁇ -D-glucopyranosyl-(1 ⁇ 6)-cellobiose and ⁇ -D-glucopyranosyl-(1 ⁇ 6)- ⁇ -D-glucopyranosyl-(1 ⁇ 6)-cellobiose were synthesized when cellobiose is used as the acceptor molecule from sucrose using alternansucrase from L. mesenterodies B-23192. However, the dextransucrase from L.
  • mesenterodies B-512 F is known primarily to transfer the D-glucose residue from sucrose to the non-reducing end 6-hydroxyl group of mono- and higher-saccharides in the presence of an acceptor molecule (Robyt, 1995; Robyt and Eklund, 1983).
  • the types of oligosaccharides synthesized by L. mesenteroides B-512 F dextransucrase apparently depends on the type of acceptor molecule.
  • dextransucrase transfers the D-glucose residue to the non-reducing end OH of maltose or isomaltose in the presence of maltose or isomaltose and transfers the D-glucose from sucrose to the reducing end D-glucose as well as the 6-OH groups of the non-reducing end in the presence of maltotriose and maltotetraose (Fu and Robyt, 1990).
  • the acceptor molecule is lactose or raffinose
  • L. mesenteroides B-512 F dextransucrase transfers D-glucose from sucrose to the OH group at C-2 of the D-glucose residue (Robyt, 1995; Robyt and Eklund, 1983).
  • Dental caries is decay of the teeth of mammals which is mainly caused by oral bacteria such as Streptococcus mutans and S. sobrinus.
  • S. mutans and S. sobrinus synthesize extracellular, water-insoluble glucans from sucrose by glucosyl transferases (Hamada and Slade, 1980).
  • the insoluble glucans become plaque on teeth and result in tooth decay. Tooth decay is promoted both by the additional bacteria that adhere to the tooth due to the glucans, but also by the increase in acid.
  • S. mutans and S. sobrinus synthesize intracellular polysaccharides as carbohydrate reserves, which can be converted to acids when dietary carbohydrates are available (Marsh, 1999).
  • mutansucrase which produces glucans with a highly branched structure from sucrose and a majority of ⁇ -(1-3)-glucosidic linkages.
  • the glucan compound produced by mutansucrase is called mutan, ⁇ -(1-3)- D -Glucan.
  • Mutan is a water-insoluble adherent, and enhances the attachment of bacteria to tooth surfaces.
  • Maltooligosylsucrose commonly known as Coupling sugar
  • palatinose also known as isomalturose
  • Antifungal agents are compounds that selectively eliminate fungal pathogens from a host, with minimal toxicity to the host. Control of fungi is crucial to prevent losses in food supplies and to decrease the fatal effects of fungal infections in patients with weakened immune systems. Studies of antifungal agents have lagged behind research on antibacterial agents. Bacteria are prokaryotic and offer numerous structural and metabolic targets that differ from those in human hosts. However, fungi are eukaryotic with the same biochemistry as mammalian cells resulting in many similarities between fungi and host cells in both cell structure and metabolism. Due to these similarities, many antifungal agents can be toxic to host cells as well as fungi. This causes toxic side effects on exposure to antifungal agents.
  • A. terreus is usually resistant to antifungal agents, including amphotericin B. Therefore, there is a real need for new effective antifungal compounds, especially against A. terreus.
  • Antifungal agents are classified into three groups: (1) antimicrobial agents affecting fungal sterol (e.g., fluconazole, itraconazole, ketoconazole, miconasol, allymine, amphotericin B, nystatin, and flucytosine), (2) agents inhibiting nucleic acids (e.g., 5-fluorocytosine), and (3) agents active against fungal cell walls (e.g., nikkomycin, demethylallosamidin, and polyxin).
  • Glucan synthesis inhibitors are compounds active against fungal cell walls.
  • Fungal cell walls are composed of ⁇ -(1,6)-glucan, mannan, or mannoprotein in the outer layers; and ⁇ -(1,3)-glucan and chitin in the inner layers.
  • Examples of fungi genera known to have glucan synthase include Candida, Aphanomyces, Paracoccidioides, Saprolegma, Aspergillus and Cordyceps .
  • glucan synthase inhibitors have been categorized into three chemical classes of compounds: (1) lipopeptides comprising cyclic hexapeptides N-linked to a fatty acyl side chain, (2) papulacandins consisting of a modified disaccharide linked to two fatty acyl chains; and (3) acidic terpenoids (Douglas, 2001; Onishi et al., 2000; Tracz 1992; Traxler et al., 1977).
  • glucan synthase inhibitors induce profound morphological changes in fungal hyphae which have been correlated with inhibition of glucan synthase (Kurtz et al., 1994; Bozzola et al., 1984; Cassone et al., 1981).
  • Inhibitors of ⁇ -(1,3)-glucan synthesis are a new therapeutic class for treating serious fungal infection.
  • the (1,3)- ⁇ -D-glucan synthase inhibitors are an effective treatment for fungal infections because these agents inhibit fungal cell wall synthesis, a target unique to lower eukaryotes (Onishi et al, 2000).
  • Echinocandins, pneumocandins, and papulacandins are known inhibitors of ⁇ -(1,3)-glucan synthase. However, they have complicated structures and are hydrophobic in nature. Although the abundance of 1,3- ⁇ - D -glucans in the cell walls formed during different stages of the A. fumigatus life cycle is not well characterized, the focus of new cell wall synthesis is the hyphae during vegetative growth (Archer, 1977; Beauvais et al., 2001: Ruiz-Herrera, 1992). Inhibition of 1,3- ⁇ -D-glucan synthesis has profound effects on cell wall structure in A. fumigatus (Kurtz et al., 1994).
  • D -gluconic acid from Pseudomonas strain AN5 has been reported to have antifungal activities against the take-all disease of wheat caused by Gaeumannomyces graminis var. tritici (Kaur et al., 2006). Some researchers have reported that cellobiose-based lipids have fungicidal activities. Complex cellobiose-lipids of yeast fungi Cryptococcus humicola and Pseudozyma fusiformata (ustilagic acid B) inhibited the growth of a number of species, important for medicine: Candida. albicans, C. glabrata, C. viswanathii, F.
  • neoformans and Clavispora lusitaninae (Kulakovskaya et al., 2007; Kulakovskaya et al., 2006). These lipids may stimulate the release of ATP from the test culture cells, indicating an increase in the permeability of plasma membrane, and resulting in cell death (Puchkov et al., 2001; Kulakovskaya et al., 2004).
  • Mimee et al. isolated flocculosin, a low molecular weight cellobiose-lipid, from the yeast-like fungus Pseudozyma flocculosa to investigate antifungal activity.
  • a cellotriose comprising three glucose molecules linked with only ⁇ -1,4, enhanced glucan synthase activity isolated from Euglena gracilis (Marechal and Goldemberg, 1964).
  • Cellobiose has been reported to be a stimulator for glucan synthase production in sugar beets (Morrow and Lucas, 1986) and in Euglena gracilis (Marechal and Goldemberg, 1964).
  • cellobiose was not found to stimulate the production of glucan synthase in S. cerevisiae (Lopez-Romero and Ruiz-Herrera, 1978) or the germinating peanut, Arachis hypogaea (Kamat et al., 1992).
  • CBO cellobio-oligosaccharides
  • Cellobio-oligosaccharides produced by dextransucrase were analyzed and shown to have a degree of polymerization (DP) ranging from 3 to 6 glucosyl groups.
  • Examples of these cellobio-oligosaccharides produced by this method include trisaccharides such as ⁇ -D-glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyranose and ⁇ -D-glucopyranosyl-(1 ⁇ 6)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyranose, and smaller amounts of tetrasaccharides, pentasaccharides, and hexasaccharides.
  • FIG. 1 illustrates the results of high performance anion exchange chromatography (HPAEC) (left) and thin layer chromatography (TLC) (right) of the cellobio-saccharides produced by dextransucrase-catalyzed transglycosylation of sucrose and cellobiose, with A and B representing trisaccharides, C representing a tetrasaccharide, D representing a pentasaccharide, and E representing a hexasaccharide.
  • HPAEC high performance anion exchange chromatography
  • TLC thin layer chromatography
  • FIGS. 2A and 2B illustrate the proposed chemical structures of two trisaccharides: ⁇ -D-glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyranose ( FIG. 2A ) and ⁇ -D-glucopyranosyl-(1 ⁇ 6)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyranose ( FIG. 2B ).
  • FIG. 3 illustrates the amount of insoluble glucan formed by bacterial mutansucrase expressed as a percent formed in the control condition under the following conditions: Control; addition of cellobiose (Cellobiose); and addition of cellobio-oligosaccharides (CBO), with bars representing the standard deviation of the mean.
  • FIG. 4 illustrates the amount of insoluble glucan on the walls of glass vials under different experimental conditions: Control; addition of 50 mM cellobiose (Cellobiose); and addition of 50 mM cellobio-oligosaccharides (CBO).
  • FIG. 5 illustrates the (1 ⁇ 3)- ⁇ -D-glucan synthase activity expressed as a percent of control activity in the presence of increasing concentrations of cellobio-oligosaccharides (0.12 g/ml; 0.24 g/ml; 0.36 g/ml; and 0.48 g/ml.
  • FIG. 6 illustrates a Lineweaver-Burk plot showing the effect on the activity of (1 ⁇ 3)- ⁇ -D-glucan synthase of the addition of increasing concentrations of cellobio-oligosaccharides (0 g/ml, 0.24 g/ml and 0.36 g/ml).
  • FIGS. 7A and 7B depict the results of Scanning Electron Microscopy (SEM) on A. terreus cells under two experimental conditions: FIG. 7A , cells grown without cellobio-oligosaccharide (arrows show sporulation); and FIG. 7B , cells grown with 50 mM cellobio-oligosaccharides.
  • FIGS. 8A-8D illustrate the in vitro growth of A. terreus in potato dextrose media in both broth (in tubes; FIGS. 8A and 8B ) and agar (in petri dishes; FIGS. 8C and 8D ) without cellobio-oligosaccharides ( FIGS. 8A and 8C ) and with 50 mM cellobio-oligosaccharides ( FIGS. 8B and 8D ):
  • Cellobio-oligosaccharides as described herein are produced by the dextransucrase-catalyzed transfer of one or more D-glucosyl moieties from sucrose to cellobiose. They are suitable for use in the inhibition of fungal growth, e.g., A. terreus , and the inhibition of dental caries.
  • the glucosyl units are added through an ⁇ (1 ⁇ 6) or ⁇ (1 ⁇ 2) linkage onto cellobiose.
  • the mixture of oligosaccharides having degrees of polymerization from 3 to 6 glucosyl groups is produced by the dextransucrase catalyzed reaction using sucrose and cellobiose.
  • oligosaccharides depends on the number of glucosyl units attached onto the cellobiose molecule (two glucosyl units of the original cellobiose molecule). For example, a single glucosyl unit attached to the cellobiose would be a trisaccharide with a DP of 3. The majority of cellobio-oligosaccharides produced were trisaccharides.
  • An example of cellobio-oligosaccharides useful as antifungal and anticariogenic agents include a mixture composed of about 66% trisaccharides with two different structures, about 13% tetrasaccharides, about 13% pentasaccharides, and about 8% hexasaccharides.
  • Cellobio-oligosaccharides are simple molecules and water soluble, thus easy for use. As with other oligosaccharides, they are expected to be non-toxic to humans. The cellobio-saccharides could be used with other antifungal agents or with other anti-cariogenic agents.
  • Leuconostoc mesenteroides B-512 FMCM a constitutive mutant from the parent B512 F for dextransucrase production, was obtained from Dr. Doman Kim (Chonnam National University, Gwangju, South Korea; and as described in Kim and Kim, 1999). The culture was grown at 30° C.
  • LM medium 0.5% (w/v) yeast extract, 0.5% (w/v) peptone, 2% (w/v) K 2 HPO 4 , 0.02% (w/v) MgSO 4 .7H 2 O, 0.001% (w/v) NaCl, 0.001% (w/v) FeSO 4 .7H 2 O, 0.001% (w/v) MnSO 4 .H 2 O, 0.013% (w/v) CaCl 2 .2H 2 O] containing 2% glucose or 2% sucrose.
  • the culture could also be maintained on glucose-LM medium containing 2% glucose and 1.5% agar at 4° C., and transferred biweekly. For growth measurement, samples of 5 ml were taken at various intervals for 48 h.
  • Bacterial growth was measured at 660 nm in a spectrophotometer using a 1 cm optical cuvette, and pH was measured directly.
  • the source of the chemicals and other agents used in the following examples were common commercial sources, usually Sigma Co. (St. Louis, Mo.), unless otherwise indicated.
  • Leuconostoc mesenteroides B-512 FMCM was sub-cultured by three successive transfers including 1 ml sucrose-LM and glucose-LM medium, 40 ml, and 1 L glucose-LM media to build sufficient volume for inoculation of the final fermentation.
  • the inoculums were 2-5% (v/v) with cultures grown for 16 h at 30° C. with shaking at 150 rpm.
  • a 400 ml culture was inoculated to 14 L of LM medium containing 2% glucose and incubated for 48 h at 30° C. The pH and agitation were not controlled during fermentation. After harvesting, cells were removed by centrifugation at 6,000 rpm ⁇ g for 30 min.
  • the cell-free culture was concentrated 10-fold using membrane filtration (100K cutoff) and washed with 2 volumes of 20 mM sodium citrate buffer, pH 5.2. Tween 80 and NaN 3 were added at concentrations of 1 mg/ml and 0.2 mg/ml to enzyme solution.
  • Dextransucrase activity was determined by incubating the enzyme with 100 mM sucrose in 20 mM sodium citrate buffer, pH 5.2 for 1 h at 30° C. and then boiling for 5 min to terminate the enzyme reaction.
  • One unit of dextransucrase activity was defined as that amount of enzyme releasing 1 ⁇ M fructose per min from 100 mM sucrose.
  • the fructose was determined by high performance liquid chromatography (HPLC) using an Aminex HPX 87K column (300 mm ⁇ 7.8 mm) and a HPLC analyzer coupled to a refractive index detector. The column was maintained at 85° C. and 0.01 M K 2 SO 4 was used as a mobile phase at a flow rate 0.6 ml/min.
  • Transglycosylation reactions were performed in 500 ml of 20 mM citrate buffer (pH 5.2) including 300 mM of sucrose, 250 mM of cellobiose, and 54 U dextransucrase at 30° C. with shaking at 150 rpm. The reaction was performed until the sucrose was depleted and then terminated by heating for 20 min at 95° C. A reaction product was centrifuged at 6,500 rpm for 45 min for the removal of insoluble polysaccharide. The soluble polysaccharide was precipitated with an equal volume of ethanol which was slowly added to the supernatant and the resulting solution stored in a refrigerator for 2 h. The precipitate was eliminated by centrifugation at 6,500 rpm for 45 min.
  • TLC thin layer chromatography
  • Oligosaccharides were also analyzed by high performance anion exchange chromatography (HPAEC) using a Dionex Carbo-Pac PA 100 column (250 ⁇ 4 mm) by gradient elution using 1 M NaOH, water and 480 mM sodium acetate at a constant flow rate of 0.5 mL/min. Oligosaccharide detection was carried out with an electrochemical detector (ED 40). The supernatant was concentrated 10-fold using a Rotary evaporator and then freeze dried.
  • HPAEC high performance anion exchange chromatography
  • the transglycosylation reaction between cellobiose and sucrose by L. mesenteroides B-512 FMCM dextransucrase produced one major product (B), several minor products (A, C, D, and E), fructose and leucrose ( FIG. 1 ).
  • the concentrations of each peak were 1.5 mg/mL for peak A, 5.5 mg/mL for peak B, 1.4 mg/mL for peak C, 1.4 mg/mL for peak D, and 0.9 mg/mL for peak E (See also, Table 1).
  • the fractions containing CBOs were recombined making a mixture that was representative of what was initially formed and was composed of about 66% trisaccharides with two different structures, about 13% tetrasaccharides, about 13% pentasaccharides, and about 8% hexasaccharides. This mixture thus contained primarily trisaccharides.
  • Mass Spectrometry Analysis Mass spectrometry data of the purified oligosaccharides were obtained from electrospray (MS-ES) measurements. The solvent was ultrapure water at 7 ⁇ l/min and detection was performed in the positive mode. The mass of the product for peaks A and B indicated 504.07 g/mol, for peak C 660.02 g/mol, for peak D 828.28, and for peak E 990.33 (Table 1). The masses of these reaction products increased over that of cellobiose by a single D-glucose residue (M.W. 162 g/mol). Therefore, peaks A and B represented compounds that were trisaccharides, peak C represented a tetrasaccharide, peak D represented a pentasaccharide, and peak E represented a hexasaccharide.
  • NMR spectra were produced using a spectrometer, operating at 500 MHz for H and 125 MHz for 13 C at 25° C. It was examined for the linkages between cellobiose and glucose from homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), rotating frame overhause effect spectroscopy (ROESY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple quantum coherence spectroscopy (HMQC) spectra.
  • COSY homonuclear correlation spectroscopy
  • TOCSY total correlation spectroscopy
  • ROESY rotating frame overhause effect spectroscopy
  • HSQC heteronuclear single quantum coherence
  • HMQC heteronuclear multiple quantum coherence spectroscopy
  • Oral bacteria were collected by a cotton swab from human teeth and streaked onto a brain heart infusion (BHI) agar containing 4% sucrose. The culture was grown at 37° C. until visible colonies of Streptococcus mutans and S. sorbrinus appeared. The colonies were grown in 1 L BHI at 37° C. with shaking at 150 rpm for 24-36 h to induce production of mutansucrase secreted outside the cell membrane. After incubation, the culture was harvested by centrifugation, and the supernatant with the mutansucrase was concentrated to 100 ml using a 30 K cut-off membrane filter. One unit of mutansucrase in the concentrated supernatant was defined as the amount of enzyme that catalyzes the formation of 1 ⁇ mol of fructose per minute at 37° C., pH 7.0, from 100 mM sucrose.
  • CBO Inhibition of Insoluble Glucan Synthesis: The inhibition of CBO on the synthesis of water insoluble glucans by oral Streptococcus species was determined.
  • CBO is the mixture of cellobio-oligosaccharides as described above in Example 1 which contains a majority of trisaccharides (>60%).
  • Streptococcus species were inoculated in 2 ⁇ BHI broth containing 1M sucrose for three treatments: one control (no additions), one with 50 mM CBO (primarily trisaccharides) and one with 50 mM cellobiose. These cultures were grown in glass vials at 37° C. for 48 h.
  • Mutansucrase in the concentrated supernatant from above was incubated in 20 mM HEPES (pH 7.0) containing 1 M sucrose in glass vials at 37° C. for 48 h under two conditions: a control (no additions) and with addition of 50 mM CBO.
  • the supernatants of individual reaction mixtures were discarded such that the insoluble glucans remained in the vial.
  • the synthesized glucans on the glass were washed with a HEPES buffer and dissolved in 0.5 N NaOH. The resulting wash was used to measure the soluble glucans.
  • the absorbance of water insoluble glucans was measured at 550 nm. For visualization, synthesized glucans were dyed with a drop of dental disclosing solution.
  • CBO insoluble glucans
  • the quantity of insoluble glucans that adhered to glass was less for the CBO mixture than the control or the cellobiose treatment.
  • the inhibitory effect of CBO was primarily the inhibition of the synthesis of glucan by mutansucrase by an acceptor reaction of glucosyltransferase, leading to termination of glucan synthesis from sucrose.
  • CBOs can be effective anti-caries ingredients in dental care products, such as toothpaste or mouth wash.
  • A. terreus A. terreus , ATCC No. 20514 (American Type Culture Collection, Manassas, Va.), was maintained on potato dextrose agar (PDA) medium for 5 to 7 days at 28° C. Conidia were collected with a cotton swab and suspended in 0.9% NaCl solution with 0.05% Tween 20. The heavy particles were allowed to settle for 2 h in cold solution. The viability was confirmed by plating serial dilutions onto PDA plates. For determination of the morphological changes, 2.5 ⁇ 10 4 conidia were inoculated into 2.9 ml of potato dextrose broth (PDB) with CBO and incubated for 10 days at 28° C. In this experiment and those following, CBO refers to the mixture of cellobio-oligosaccharides as described above in Example 1 which contains a majority of trisaccharides (>60%).
  • PDA potato dextrose agar
  • Cell breakage was performed using 20 cycles (1 min each) of vortexing with pre-chilled glass beads in chilled extraction buffer containing 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; pH 7.2), 1 mM EDTA, 1 mM dithiothreitol (DTT), 10% glycerol, 1 ⁇ g of leupeptin per ml, and 10 ⁇ M GTPrS at approximately 5 ml of buffer per cell. Cells were cooled for 5 min on ice between cycles. The homogenate was centrifuged at 1,500 ⁇ g for 10 min to remove cell debris.
  • HEPES N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
  • Glucan synthase activity was determined by the modification of a fluorescence method, as described in Shedletzky et al. (1997).
  • the assay mixture (150 ⁇ l) contained 27 mM HEPES (pH 7.2), 7 ⁇ M GTP, 1.3 mM EDTA, 0.17% Brij 35, 2.2% glycerol, 0.7 mM UDP-Glc, and isolated GS enzyme (0.83 ⁇ g/ ⁇ l).
  • CBO at concentrations from about 0.12 to about 0.63 ⁇ g was added to the desired mixture. Reactions were started by addition of GS, incubated at 22° C. for 105 min, and terminated by an addition of 10 ⁇ l of 6 N NaOH.
  • glucans produced were solubilized in a water bath at 80° C. for 30 min followed by an addition of 20 ⁇ l of a 4:1 diluted Sirofluor, a chemically defined fluorochrome from aniline blue.
  • the mixtures were further incubated for 50 min at 22° C., and measured with a fluorescence spectrophotometer (FluoroLog, Horiba Jobin Yvon, Edison, N.J.) at an excitation wavelength of 390 nm and an emission wavelength of 455 nm.
  • Standard curves were constructed using various concentrations of yeast glucan, dissolved in 300 ⁇ l of 1 N NaOH by heating 30 min at 80° C., containing the same components as the reaction mixtures except for enzyme.
  • Relative Activity is the percent of 1,3- ⁇ - D -glucan synthase activity (GS) at a test concentration of cellobio-oligosaccharides (CBO) as compared to the GS activity in the control (no CBO).
  • GS activity 0.7 mM UDP-G was reacted with 0.83 ⁇ g/ ⁇ l GS in 27 mM HEPES (pH 7.2) containing 7 ⁇ M GTP, 1.3 mM EDTA, 0.17% Brij 35, and 2.2% glycerol with the addition of 0, 0.12, 0.24, 0.36, and 0.48 g/ml CBO (primarily trisaccharides) at 22° C. for 105 min.
  • a SirofluorTM binding with 1,3- ⁇ - D -glucans was then conducted as described above. The fluorescence was measured at an excitation wavelength of 390 nm, and an emission wavelength of 455 nm.
  • the role of CBO on glucan synthase was further evaluated by a kinetic study over a range of concentrations (0.05 to 8 mM) of UDP-glucose with CBO added at concentrations of 0, 0.24, and 0.36 g/ml.
  • the assay mixtures contained 27 mM HEPES (pH 7.2), 7 ⁇ M GTP, 1.3 mM EDTA, 0.17% Brij 35, and 2.2% glycerol, varying concentration of UDP-G (0.05, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 ⁇ g/ ⁇ l), and 0.83 ⁇ g/ ⁇ l 1,3- ⁇ - D -glucan synthase (GS).
  • the reaction was allowed to react for 105 min at 22° C. GS activity was also measured with the addition of 0, 0.24, and 0.36 g/ml cellobio-oligosaccharides.
  • a SirofluorTM binding with 1,3- ⁇ - D -glucans was then conducted as described above. The fluorescence was measured using an excitation wavelength of 390 nm and emission wavelength of 455 nm. The reaction velocity was calculated, and a Lineweaver-Burk plot of 1/[substrate] and 1/velocity at three oligosaccharide concentrations was plotted. These results are shown in FIG. 6 . The non-parallel lines which converge at x ⁇ 0 and y>0 are consistent with a mixed type of inhibition.
  • Morphological Changes in Fungi due to CBO Morphologic changes in fungi due to the addition of CBO to growth media were monitored using scanning electron microscopy (SEM).
  • Conidia (3.0 ⁇ 10 4 ) were inoculated in PDB (potato dextrose broth) and incubated at 28° C. After incubation for 16 h, 50 mM CBO was added to one inoculated tube, and water added to a second tube as a control. The tubes were further incubated for two days at 28° C. For SEM, the incubated cultures were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at 4° C. for 2 hs.
  • specimens were post-fixed for 2 h with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) at 4° C. Samples were dehydrated in graded acetone, freeze-dried in t-butyl alcohol, and then sputter-coated with palladium-gold.
  • Hyphae of A. terreus showed distinct structural differences between control and CBO-treated cultures (50 mM CBO) ( FIGS. 7A and 7B ).
  • the bud scar rings are found in several hyphae tips on the control ( FIG. 7A ) but none on CBO treated A. terreus ( FIG. 7B ).
  • the hyphae of CBO-treated A. terreus fail to bud, and the population does not increase.
  • the average width of hyphae was different between the two cultures when 20 hyphae were randomly selected and measured.
  • the average width of twenty hyphae in CBO-treated A. terreus (3.4 ⁇ m) was 1.35 fold larger than the width in the control (2.5 ⁇ m).
  • the cells grew with swollen hyphae, indicating inhibition of glucan synthesis.
  • the term “effective amount” as used herein refers to an amount of cellobio-oligosaccharides sufficient either to inhibit production of glucans on teeth or to inhibit the growth of fungi to a statistically significant degree (p ⁇ 0.05).
  • the term “effective amount” therefore includes, for example, an amount sufficient to reduce glucan production in the mouth or reduce fungal growth by at least 50%, and more preferably by at least 90%.
  • the dosage ranges are those that produce the desired effect. Generally, the dosage will vary with the type of fungi, age, weight, or condition. A person of ordinary skill in the art, given the teachings of the present specification, may readily determine suitable dosage ranges. The dosage can be adjusted by the individual physician in the event of any contraindications.
  • the effectiveness of treatment can be determined by monitoring the extent of fungal or glucan reduction by methods well known to those in the field.
  • the cellobio-oligosaccharides can be applied in pharmaceutically acceptable carriers known in the art.
  • the application will be oral to reduce the glucans, and will be oral, by aspiration, or by injection for an antifungal agent.
  • the cellobio-oligosaccharides can be a mixture of compounds with degrees of polymerization from DP 3 to 6, or can be cellobio-oligosaccharides with a single DP, preferably 3.
  • One example of an effective mixture is one in which the majority of cellobio-oligosaccharides are trisaccharides.
  • cellobio-oligosaccharides useful as antifungal and anticariogenic agents include a mixture composed of about 66% trisaccharides with two different structures, about 13% tetrasaccharides, about 13% pentasaccharides, and about 8% hexasaccharides.
  • the cellobio-oligosaccharides may be administered to a patient by any suitable means, including oral, parenteral, subcutaneous, intrapulmonary, topically, and intranasal administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or intravitreal administration.
  • the cellobio-oligosaccharides may also be administered orally in the form of capsules, powders, or granules.
  • Pharmaceutically acceptable carrier preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • the cellobio-oligosaccharides may be mixed with excipients that are pharmaceutically acceptable and are compatible with the active ingredient.
  • Suitable excipients include water, saline, dextrose, and glycerol, or combinations thereof.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like.
  • the cellobio-oligosaccharides could also be mixed with dental care products, e.g., toothpaste or mouthwash. They could also be mixed with other antifungal agents, e.g., echinocandins, pneumocandins, papulacandinsfluconazole, itraconazole, ketoconazole, miconasol, allymine, amphotericin B, nystatin, flucytosine), 5-fluorocytosine, nikkomycin, demethylallosamidin, polyxins, flocculosin, and ⁇ -gluconolactone.
  • dental care products e.g., toothpaste or mouthwash.
  • antifungal agents e.g., echinocandins, pneumocandins, papulacandinsfluconazole, itraconazole, ketoconazole, miconasol, allymine, amphotericin B, nystatin, flucytosine), 5-fluorocytos

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US10428362B2 (en) 2014-02-27 2019-10-01 E. I. Du Pont De Nemours And Company Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes
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US10428362B2 (en) 2014-02-27 2019-10-01 E. I. Du Pont De Nemours And Company Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes
US10633683B2 (en) 2015-04-03 2020-04-28 Dupont Industrial Biosciences Usa, Llc Gelling dextran ethers
US10787524B2 (en) 2015-04-03 2020-09-29 Dupont Industrial Biosciences Usa, Llc Oxidized dextran
CN107281013A (zh) * 2017-07-28 2017-10-24 鹰仕达（中国）有限公司 一种去除牙渍抑制牙菌斑牙膏
CN107362112A (zh) * 2017-07-28 2017-11-21 鹰仕达（中国）有限公司 一种护龈愈合牙膏
CN107468553A (zh) * 2017-07-28 2017-12-15 鹰仕达（中国）有限公司 一种防龋齿牙膏
CN107485584A (zh) * 2017-07-28 2017-12-19 鹰仕达（中国）有限公司 一种口腔护理组合物

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